Infectious diseases

They are clinically evident diseases with the potential of

transmission from one person or species to another.
They result from the presence of pathogenic
microorganisms (very small organisms that are
invisible to the naked eye) that are able to cause disease
in human beings.
Pathogenic micro-organisms include bacteria, viruses,
fungi, protozoa, and multicellular parasites.
Practical microbiology sessions focus on the diagnosis of
these infectious agents.
 Laboratory diagnosis of
infectious diseases

Direct method Indirect (serologic) method

Detection of antibodies
against the microorganism in
Detection of: the patient’s serum.
 microorganisms,
 their structural components
 their products
in specimens collected from the patient (e.g.
urine, blood, sputum, CSF……etc).
 I) DIRECT METHOD
1)Specimen Collection
A ’good quality’ clinical specimen.

2) Microscopic Examination:
usually before further processing of specimens

3) Microbial Detection:
a) Culture technique:
b) Non-culture technique
I) DIRECT METHOD
1) Specimen Collection
A ’good quality’ clinical specimen

 Collecting specimens before the start of antibiotics.

 Choosing the appropriate specimen (representing the infection

site).

 Using sterile containers and avoiding contaminating the

specimen.

 Transporting the specimen properly to the lab. as early as

possible.
Specimen Collection
I) DIRECT METHOD
2) Microscopic Examination:
usually before further processing of specimens
 stained /unstained (wet)preparations
 different types of microscopes

Staphylococci in pus
I) DIRECT METHOD
3) Microbial Detection:
a) Culture Technique:
 Isolation of the organism in pure culture
→inoculating the specimen onto appropriate artificial culture media

followed by
 Identification of the isolate by e.g. :
 microscopic examination
 biochemical reactions
 reaction with specific antibody (serologic identification of the organism)
 DNA probes
Which of these approaches is used and in what sequence depends upon the type of specimen and
organism.

 Antibiotic sensitivity
After growing the organism in pure culture.
I) DIRECT METHOD
3) Microbial Detection:
b) Non-Culture Technique:

I. Identification of a specific microbial antigen such as :

 a structural component (e.g. cell wall antigen, capsular polysaccharide…etc) or

 a product (e.g. an exotoxin)
by reacting with specific antibody

OR:

II. Identification of a specific gene sequence

(i.e. nucleic acid of the organism)

by the application of different molecular methods (e.g. PCR, DNA probe).

I) DIRECT METHOD

 yield more rapid results (minutes or hours)

(do not depend on growth and multiplication of the organism)
However, antimicrobial susceptibility cannot be determined
(although the presence of resistance genes can be determined by
molecular methods).

 Non-culture techniques are mainly applied if:

 a rapid diagnosis is needed

 The microorganism cannot be cultured on artificial
media
 A slowly growing micro-organism
 II) INDIRECT (SEROLOGIC)
METHOD
 Serologic diagnosis of infectious diseases involves the use of known
microbial antigens to detect antibodies against the
microorganism in the patient’s serum.

current (active) infection is diagnosed by the

detection of one of the following

 specific IgM antibodies

 rising titre of specific IgG antibodies (4-fold or greater rise)
 a single high titre of IgG antibodies in certain diseases

N.B. Skin tests based on cell-mediated hypersensitivity may also

help in the diagnosis of certain diseases.
Skin Test
 Diagnosis
Diagnosisof
ofinfectious
infectiousdiseases
diseases

DIRECT
DIRECTMETHOD
METHOD INDIRECT
INDIRECTMETHOD
METHOD
(SEROLOGICAL)
(SEROLOGICAL)
Specimen
Specimen

Microscopical
Microscopicalex.
ex. Detection
Detectionof ofantibodies
antibodiesin
inserum
serum
••IgM
IgM
••rising
risingtitre
titreof
ofIgG
IgG
Culture
Culturetechnique
technique Non-culture
Non-culturetechnique
technique

Isolation
Isolationon
onculture
culturemedia
media ••detection
detectionofofspecific
specificantigen
antigen
(serology)
(serology)
.Identification
.Identificatione.ge.g •• detection
detectionof
ofspecific
specificgene
gene
••microscopical.
microscopical.ex.
ex. sequence
sequence(mol.
(mol.tech.)
tech.)
••bioch.
bioch.Reactions
Reactions
••DNA
DNAprobe
probe
••serology
serology Antibiotic
Antibioticsensitivity
sensitivity
Microscopy
Light Microscope
 Stained Preparations Unstained preparations

Bacterial morphology Motility

 Bacterial Morphology
Size
Shape
Special arrangement
Staining affinity
Spore formation
Capsule formation
Motility
Bacterial Shape
 Bacterial arrangement
Chains.

Pairs (diploids).

Clusters (group).

No special arrangement.

Bacterial arrangement
Cocci

Pairs Chains Irregular Clusters

22
Bacterial arrangement
Bacilli

Pairs chains No special arrangement

23
 Spore formation
 Morphological characters of bacterial spores:
* Shape.
* Position.
* Staining.
Bacterial spores
Bacterial capsule
 Staining of Bacteria
Bacteria cells are almost colorless and
transparent

A staining technique is often applied to the

cells to color them →
Their shape and size can be easily
determined under the microscope.

28
Smear preparation

S Fixation

30
Smearing out of the sample
 Types of Stains
1- simple stain:
Single basic dye e.g. Methylene blue
All bacteria take the color of the dye

2- Differential stain:
Two dyes
separated by a decolorizing agent
e.g. Gram stain
& Ziehl-Neelsen stain
3- Special stain: e.g. Fontana stain
 Differential staining
Principles of differential stain
* Application of the main stain.
• Decolourization.
*Application of the counter-stain.

e.g. Gram stain & Ziehl-Neelsen

stain
1. Gram Stain
 Components

Primary Stain: Methyl violet + Gram’s iodine

Decolourizing agent: 95% ethyl alcohol

Counter stain: dil. Carbol fuchsin

 Principle
Primary Stain Methyl violet + Gram’s iodine →
All Violet

Decolourizing agent: 95% ethyl alcohol

Not decolourized( violet ) decolourized ( colourless )

( Gram + ve) (Gram –ve )

Counter stain: dil. Carbol fuchsin→ colourless→pink(Gram -ve )

Gram Staining Technique
Gm+ve cocci & G-ve bacilli
2. Ziehl-Neelsen
Stain
Mycobacterium
A 3rd type of cell envelope (high lipids content of cell
wall)

Not readily stainable with ordinary stains

.
A strong stain e.g., concentrated carbol fucsin + heat.

Resist decolorization by strong mineral acids or

acid-alcohol →

Acid-fast.
 Components

Primary Stain: Conc. Carbol fucshin

Decolourizing agent: 20% H2SO4 OR

3% HCL in alcohol

Counter stain: methylene blue

 Principle
Primary Stain Conc. Carbol fucshin → All Red

Decolourizing agent: 20% H2SO4

OR 3% HCL in alcohol

Not decolourized ( red ) decolourized ( colourless )

( acid-fast) (non acid-fast)

Counter stain: methylene blue → Colourless →blue

(non acid-fast)
Ziehl-Neelsen Stain Technique
1 2 3

4 5 6

7
Questions
5.Acid fast bacilli stained by Z-N stain appear:
a) Violet
b) Blue
c) Red
d) Colourless
e) brown

6.The steps of Gram’s stain is as follows:

a) methyl violet / ethyl alcohol / dil. carbol fuchsin / iodine
b) methyl violet / ethyl alcohol / iodine / dil. carbol fuchsin
c) dil. carbol fuchsin / methyl violet / ethyl alcohol / iodine
d)dil. carbol fuchsin / iodine / methyl violet / ethyl alcohol
e)methyl violet / iodine /alcohol / dil. carbol fuchsin